Nonpeptide insulin receptor agonists

ABSTRACT

Modulation of the activity of the insulin receptor, enhancement of glucose uptake by cells, and other effects significant in the control and management of diabetes are accomplished using compounds of the formula ##STR1## wherein each Ar is independently an aromatic moiety; each A is independently a proton-accepting substituent; 
     each R is independently a noninterfering substituent; 
     m is 0 or 1; 
     n is 4-6; and 
     each linker is independently an isostere of --CH 2  --, --CH═CH-- or --NCHO--. Compounds in the genus of Formula (1) can also be used for structure activity studies to identify features responsible for the relevant activities.

TECHNICAL FIELD

The invention relates to the substitution of nonpeptide compounds forpeptide ligands that activate hormone receptors. More specifically, itconcerns simple nonpeptide compounds that behave as agonists for theinsulin receptor and enhance the effect of insulin on this receptor.

BACKGROUND ART

Among the many functions performed by peptides and proteins inmetabolism is the ability to stimulate receptors at cell surfaces toeffect intracellular consequences important in maintenance anddevelopment of the organism. Peptide and protein hormones interact withreceptors specific for them so that the activity of the hormone is felton designated cells exhibiting these receptors. The insulin receptor ispresent on virtually all cells and at high concentrations on the cellsof the liver, skeletal muscles, and adipose tissue. Stimulation of theinsulin receptor with insulin is an essential element in carbohydratemetabolism and storage.

Diabetics either lack sufficient endogenous secretion of the insulinhormone (Type I) or have an insulin receptor-mediated signaling pathwaythat is to some degree resistant to endogenous or exogenous insulin,either through primary or post-translational structural changes, reducednumbers or poor coupling among signaling components (Type II). All TypeI diabetics, and many Type II subjects as well, must utilize injectionto obtain enhanced activity of the extant insulin receptors, sinceendogenous insulin can at present be replaced only with an alternativesupply of insulin itself, previously isolated from native sources, andnow recombinantly produced. While the recombinant production of insulinpermits a less immunogenic form to be provided and assures a reliablesupply of needed quantities, the necessity to administer the hormone byinjection remains, due to the instability of peptides and proteins inthe digestive tract. It has long been the goal to substitute for peptideligands, including insulin, small molecules which are not digested andcan be absorbed directly into the bloodstream. However, to date,nonpeptide substances which can exert the effect of insulin on itsreceptor have eluded discovery.

There have been many instances in which nonpeptide materials have beenused to inhibit enzymes whose native substrates are peptides. Forexample, Brinkworth, R. I. et al. Biochem Biophys Res Comm (1992)188:624-630 describe the inhibition of HIV-1 proteinase by various aryldisulfonates. The ability of triazine dyes to bind NADH oxidase fromThermus thermophilus was studied by Kirchberger, J. et al J Chromatog A(1994) 668:153-164.

It has also been shown that certain nonpeptide components enhance theagonist properties of a peptide hormone, The ability of certainthiazolidinediones such as pioglitazone to enhance adipocytedifferentiation by stimulating the effect of insulin has been describedby, for example, Kletzien, R. F. et al. J Mol Pharmacol (1992)41:393-398. These represent a class of potential antidiabetic compoundsthat act at an unknown site downstream from the insulin receptor itselfand enhance the response of target tissues to insulin. Kobayashi, M.Diabetes (1992) 41:476-483. It is now known that most of thethiazolidinediones bind to PPARγ thus triggering certain nuclear eventsthat may result in enhanced sensitivity of the target cells to insulin.However, the complete mechanism is still unresolved.

In any event, it has not as yet been possible to utilize simplemolecules to provide the effect of a peptide hormone by stimulatingreceptor activity independently of the peptide hormone binding site.

It has now been found that several aryl di- or polysulfonate compoundswhich share certain common structural features are able to effectstimulation of the insulin receptor to activate the autophosphorylationactivity required for signal transduction. The availability of thesecompounds permits construction of assays and comparative procedures forevaluating additional candidate compounds as well as the design andsynthesis of therapeutics for primary treatment of insulin resistanceand diabetics with the appropriate structural features.

DISCLOSURE OF THE INVENTION

The invention takes advantage of the behavior of, and informationprovided by, certain compounds, whose synthesis is straightforward, inorder to conduct assays for the ability of candidate small molecules toactivate the insulin receptor and to design these candidates. The methodof identifying a primary member of this group, TER12 and of obtainingthe remaining members is described below. These small moleculesrepresent the first instance of direct agonist activity on the insulinreceptor by a nonpeptide. Compounds identified in this way are useful inthe control and management of diabetes in suitable subjects.

Thus, the invention is directed to methods to modulate the kinaseactivity of the insulin receptor or the kinase portion thereof; topotentiate insulin activation of the insulin receptor; to potentiateglucose uptake stimulation by insulin; to lower blood glucose; and tostimulate glucose uptake per se in cells by use of compounds having theformula ##STR2## wherein each Ar is independently an aromatic moiety;each A is independently a proton-accepting substituent;

each R is independently a noninterfering substituent;

m is 0 or 1;

n is 4-6; and

each linker is independently an isostere of --CH₂ --, --CH═CH-- or--NHCO--.

These compounds are thus useful in regulating the glucose metabolism ofmammalian subjects which are afflicted with diabetes.

In another aspect, the invention is directed to a method to screencandidate compounds for ability to activate the insulin receptor. Themethod comprises first obtaining a fingerprint of each candidate withrespect to a reference panel and obtaining a fingerprint of an activatorwhich is Component A with respect to the same reference panel. Then thefingerprint of each candidate is compared with that of Component A.Successful candidates are compounds whose fingerprints resemble that ofComponent A. ##STR3##

In another aspect, the invention relates to a method to design andsynthesize a molecule that exhibits agonist activity or insulin agoniststimulating activity with respect to the insulin receptor. This methodcomprises assessing an activator identified as Component A structuralfeatures which correlate with said activities. Compounds containingthese structural features are designed and synthesized.

In still another aspect, the invention provides an alternative method toidentify a candidate compound which will activate the insulin receptor.This method comprises contacting a sample containing at least the kinaseportion of the insulin receptor with an activator which is Component Ain the presence and absence of said candidate.

The binding of said activator is then measured in the presence andabsence of said candidate. For a successful candidate, the binding ofactivator is diminished in its presence as compared to its absence.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic diagram of the insulin receptor and itsactivation by insulin.

FIGS. 2A-2D show the structures of several compounds relevant to theinvention which activate the insulin receptor. FIG. 2A shows thestructure of TER12, Cibacron Brilliant Red 3BA; FIG. 2B shows thestructure of TER3938, Direct Yellow 27; FIG. 2C shows the structure ofTER3935; FIG. 2D shows the structure of TER16998.

FIG. 3 shows the pathway for synthesis of TER12.

FIG. 4 shows the effect of Component A on insulin-induced uptake ofglucose by adipocytes.

FIG. 5 shows the synthetic pathway for TER16998.

FIG. 6 shows the effect of TER16998, alone and in combination withinsulin, on autophosphorylation of the IR receptor.

FIG. 7 shows the effect of TER16998 on insulin-induced glucose uptake inadipocytes.

FIG. 8 shows the effect of TER16998 on blood glucose levels in adiabetic mouse model.

FIG. 9 shows the synthetic method for preparation of compounds similarto Component A.

MODES OF CARRYING OUT THE INVENTION

The structure of the insulin receptor and some aspects of its mode ofaction as currently understood, are illustrated in FIG. 1. The receptorconsists of four separate subunits consisting of two identical α and twoidentical β chains. The two β chains contain a cross-membrane domain;the α portions are in the extracellular domain and accommodate thebinding of insulin. The illustration in FIG. 1 shows insulin bound tothe receptor. The β subunits contain a tyrosine kinase activity, shownas the white inserts into the subunits and the kinase of one β subuniteffects the phosphorylation of the complementary β subunit as shown; thereceptor illustrated in FIG. 1 is in its activated form when thetyrosine residues (Y) are phosphorylated. The β subunits also containATP binding sites. The insulin-stimulated phosphorylation of thereceptor itself is required for subsequent activity and thusdemonstration of the ability of a compound to effect phosphorylation ofthe β subunits provides a means to assay activation of the receptor.

The invention, in general, is directed to methods to regulate and managesubjects with diabetes by virtue of administering compounds which affectthe activity of the insulin receptor. Without intending to be bound byany theory, it is believed that the compounds useful in the methods ofthe invention act directly on the kinase function of the receptor and donot necessarily compete with insulin for binding at the insulin-bindingsite, nor do they effect activation of the receptor by a mechanismsimilar to that exhibited by insulin. The compounds of the invention areable directly to activate the kinase of the receptor toautophosphorylate, to potentiate the effect of insulin on the receptor,to activate the kinase function of the receptor in phosphorylatingexogenous substrates, to effect the increased uptake of glucose byadipocytes and insulin receptor-bearing cells in general, and to lowerblood glucose levels in diabetic subjects.

The compounds of the invention are generally of the formula ##STR4##wherein each Ar is independently an aromatic moiety; each A isindependently a proton-accepting substituent;

each R is independently a noninterfering substituent;

m is 0 or 1;

n is 4-6; and

each linker is independently an isostere of --CH₂ --, --CH═CH--, or--NHCO--.

In the compounds of Formula (1), the proton-accepting substituentsrepresented by "A" may be anionic or may be sufficiently nucleophilic toaccept a proton at physiological pH. Particularly preferred embodimentsof A include --SO₃ X, OP(OX)₃ and --COOX where X is a hydrogen atom or acation depending on pH. Suitable cations include inorganic cations suchas sodium, potassium, calcium and the like or may be organic cationssuch as those provided by organic bases, for example, caffeine. Alsoincluded in embodiments of A are amino substituents including primary,secondary, and tertiary amines. Typical bioisosteres of anionic ligandssuch as tetrazole rings, even when they are not charged.

The aromatic moieties represented by Ar are monocyclic or bicyclicaromatic systems such as benzene or naphthalene or contain one or moreheteroatoms selected from the group consisting of O, S and N. Thus,included among the aromatic systems are benzothiazoles, quinolines,pyridine, and the like. Particularly preferred are naphthylene residues.

The noninterfering substituents on the naphthyl moieties in Formula (1)may or may not be present--i.e., each m is independently 0 or 1. Theposition of R is arbitrary in each case; preferred embodiments of Rinclude substituted or unsubstituted hydrocarbyl moieties, whetherstraight-chain, branched or cyclic and whether aromatic or nonaromatic.Among these are included but not limited to alkyl substituents of 1-6C,alkenyl substituents of 1-6C, and alkyl or alkenyl substituents whereinthe carbon chain is interrupted by one or more heteroatoms such as O, Nor S. Substituents may also be of the formula --OR', --NR'₂ and --SR',wherein R' is H or is R as defined above. Particularly preferredembodiments include alkyl (1-6C).

In the compounds of Formula (1), each linker is independently anisostere of --CH═CH-- such as --N═N--, --CH═N-- or --CH₂ CH₂ -- --NHCH₂,or --CH₂ -- such as NH or O, or of --NHCO-- such as --OCO-- or --COO--.General methods for forming all of these linkages between aromaticsystems are well known in the art.

Particularly preferred compound of Formula (1) are those wherein each Ris alkyl (1-6C). Also preferred are compounds of Formula (1) whereineach m is 0.

Also preferred are embodiments wherein the compound of Formula (1) is##STR5## wherein n is 4-6 and A is as defined in claim 1.

Particularly preferred is the compound shown as Component A.

In addition to protocols to control diabetes in relevant subjects, theinvention is directed to methods to identify additional compounds whichare useful in these protocols. In general, such methods includeidentifying an active compound by a screening process that utilizes aset of maximally diverse candidate compounds. This method comprises, ina preferred embodiment, contacting each member of a set of maximallydiverse candidate compounds with said receptor or kinase portionthereof; detecting the presence or absence of tyrosine phosphate on thereceptor or kinase portion contacted with each set member; andidentifying as a successful candidate at least one member of the setwherein an increased amount of tyrosine phosphate is detected in thereceptor or kinase with which it was contacted, relative to untreatedreceptor.

In addition, once a compound with at least moderate ability to activatethe kinase activity of insulin receptor has been identified, additionalcompounds can be identified by comparing the properties of thecandidates with those of compounds having known activity. Oneparticularly useful property to compare is the affinity fingerprint ofthe compound against a reference panel of proteins which provide a firstapproximation of the binding modes of all proteins. This is described inU.S. Pat. No. 5,587,293, incorporated herein by reference. Further,analysis of compounds shown to activate the insulin receptor kinaseusing standard structure activity analysis will result in additionalcompounds which behave as activators. Compounds identified as activatorsof the receptor in any of the foregoing three ways can further be usedin competitive analyses with respect to additional candidate compounds,particularly if radiolabeled. Receptor mutagenesis and photoaffinityanalogs may also be used to identify the receptor site binding thecompounds, for use in rational drug design.

The three primary methods of identification of compounds with thedesired IR kinase modulating activity are illustrated below.

The activator compounds are able to stimulate the phosphorylationcatalyzed by IR kinase alone, i.e., to behave as agonists with respectto the receptor and/or are able to enhance the ability of insulin toeffect phosphorylation of the receptor. Either of these effects can beconsidered an activation of the insulin receptor. Thus, by "activating"the insulin receptor is meant either the ability to behave as an agonistor the ability to enhance the stimulation by insulin or other agonistsof the receptor activity. Both of these effects can be evidenced byautophosphorylation of the receptor.

The compounds of the invention evidently do not interact with thereceptor at the native insulin binding site, but rather at a sitelocated on the kinase portion of the receptor. Thus, these compoundsdefine a newly discovered activation site for this receptor. Thispermits not only the identification of compounds with similar activitiesthrough competitive binding assays with the identified compound (whichdirectly identifies compounds which interact with the same site), butalso permits these assays to be conducted with forms of the receptorcontaining only the kinase portions.

Identification of Desired Activator Compounds--Maximally DiverseLibraries

A traditional approach to the identification of a substance which bindsto a desired target is through simple trial and error. Done in a randomfashion with each compound available in a chemical library, thisapproach is labor- and time-intensive for a complex assay such asautophosphorylation of IRk. As previously described by two of thepresent inventors, the number of trials can be minimized by testing onlyrepresentative substances across the totality of chemical space. Thus,only members of a maximally diverse set of candidate compounds is testedin the trial and error procedure. One way to construct a maximallydiverse set of candidates is described in U.S. Pat. 5,340,474,incorporated herein by reference. In this approach, the members of theset are synthesized from subunits wherein the subunits are chosen toresult in a systematic variation of parameters, such as hydrophobicity,hydrophobic moment and the like, which determine the position of theresulting compound in chemical space. Alternatively, the maximallydiverse set of test compounds can be obtained by manipulating affinityfingerprints. The fingerprints of multiple members of a compound librarycan be searched and clustered to obtain groups with similarfingerprints. Each group represents a different general location inchemical space. Iterations of this process may be continued until amanageable number of compounds representing the totality of chemicalspace are identified. Thus, from a cluster of, for example, fingerprintsfor 100 compounds with similar properties, only one of these 100compounds need be included in the group of compounds to be tested forbinding to the target. This is described in U.S. Pat. No. 5,587,293,cited above.

In the present application, a library containing the fingerprints of10,000 compounds obtained against a panel of 18 reference proteins wassorted as described, i.e., clusters of fingerprints with similarcharacteristics were grouped to select 50 representative compounds as a"training set." Each of these 50 representative compounds was testedwith respect to the insulin receptor. A sample believed to consist onlyof TER12 shown in FIG. 2A, whose fingerprint did not group and was not amember of a cluster was the only tested compound that was successful inactivating this receptor using the assay set forth in Example 1.Although it was later found that the component of the tested sample thathad the structure of TER12 was not as active in this assay as aComponent A, present at lower concentration, the structural similaritiesof TER12 and the actual active component are evident.

Fingerprint Comparison

Chemical catalogs were searched for compounds with structural featuressimilar to those of TER12 or which when tested had fingerprints showingessential matching features. Of 42 compounds identified as havingsimilar substructures, four showed activity with respect to thereceptor; among these was a sample containing TER3938 shown in FIG. 2B,although most of the activity was later shown to be due to Component Apresent at lower concentration.

The fingerprints of these compounds identified with respect to astandard reference panel (further described hereinbelow) can be used asstandards for comparison to fingerprints of candidate compounds likelyto have the same activities.

U.S. Pat. No. 5,217,869 and 5,300,425, the contents of which areincorporated herein by reference, describe techniques for identifyingcompounds with similar properties by comparing their fingerprints.Briefly, a fingerprint for a single compound (which characterizes it) isobtained by testing the binding or reactivity of the compound withrespect to a reference panel of substances which may, for instance, beantibodies or other substances which exhibit varied degrees ofreactivity with respect to most compounds. The reference panels arechosen so that they represent virtually the totality of chemicalspace--i.e., a set of substances so varied in its spatial and chargecontours that the ability to react with any other substance is containedat least somewhere within the panel. Each compound reacted with thepanel, then, yields a characteristic pattern of reactivities which couldbe considered a fingerprint. Compounds which exhibit similarfingerprints exhibit similar patterns of reactivity and properties.Thus, if a target receptor is known to bind to a specific ligand, onecan identify a compound which also binds to the receptor by choosing acompound whose fingerprint is similar to that of the known ligand. Here,the fingerprints of candidates from, for example, libraries of compoundsare compared to the corresponding fingerprints of TER12, TER3938 orpreferably Component A.

Structure Activity Relationships

Assessment of the structural features of an individual active compound,especially those that are shared by several active compounds, incontrast, for example, to the compounds which do not activate theinsulin receptor, permits the design of suitable candidates forsynthesis and testing. Methods for such analysis and identification ofsuch structural features are known in the art. See, for example, Nesnow,S. et al. J Toxicol Environ Health (1988) 24:499-513, which describesthe assignment of structural features among a group of 36 arylazo dyesas related to their ability to be reduced by rat liver microsomalazoreductase.

Competitive Binding

Once activators of the insulin receptor such as Component A (or anyreceptor) have been identified either by screening a maximally diverselibrary or by using the results of such screening to comparefingerprints and selecting fingerprints similar to those of thesuccessful compounds, or by structure activity analysis of one or moreactivator compounds in comparison to those which are inactive, thesesuccessful activator compounds can be used to screen for additionalsubstances which behave using similar mechanisms by competitive bindingassays wherein the activator compounds, for example labeled withradioisotopes, fluorescent labels, enzyme labels, and the like, arecontacted with the insulin receptor or the insulin receptor kinase inthe presence and absence of candidate insulin receptor activatorcompounds. The amount of label bound to the receptor or to its kinaseportion is measured in the presence and absence of the candidate; anincreased level of label binding in the absence, as opposed to thepresence of the candidate indicates that the candidate successfullycompetes for the newly defined binding site and is a successfulcandidate as an activator of the receptor.

The following examples are intended to illustrate but not to limit theinvention.

EXAMPLE 1 Apparent Effect of TER12 on Insulin Receptor KinaseAutophosphorylation

A. This assay is a modified form of that described in Hagino, H. et al.Diabetes (1994) 43:274-280. Briefly, human insulin receptors (hIR) werepartially purified from placental extracts or from cell line IM-9. Thepartially purified hIRs were captured into microplate wells byincubating them for 90 minutes with wells coated with a monoclonalantibody to hIR. The wells were then treated with various dose levels ofinsulin and/or test compounds for 15 minutes at room temperature; ATP(10 μM) was then added to permit kinase activity to proceed. After 60minutes, the wells were washed, and then treated for 60 minutes withbiotinylated antibody directed against phosphotyrosine (PY-20) andunbound materials again washed away. The wells were then incubated witha conventional streptavidin peroxidase system for 30 minutes to assessthe level of phosphorylated tyrosine.

When tested in this assay, insulin gave a dose response curve showing anEC₅₀ of about 0.3 nM and a maximal activity at about 100 nM. The EC₅₀ issimilar to that obtained for binding of labeled insulin to various cellsand tissues.

Two sets of compounds of 50 members each, selected to be maximallydiverse, as defined in U.S. Pat. No. 5,300,425 and incorporated hereinby reference, were screened in the foregoing assay. Of these 100compounds, only a sample composed mainly of TER12 (see FIG. 2A) showedapparent agonist activity. In the absence of insulin, 20 μM of thissample stimulated autophosphorylation over five-fold (0.3 nM insulinstimulates phosphorylation approximately to this extent). Thus, theactivity of insulin at approximately 0.3 nM is roughly equivalent tothat shown by this sample at approximately 20 μM and a component of thissample shows the ability directly to stimulate autophosphorylation.

In addition, the sample enhanced the ability of insulin to stimulateautophosphorylation. The addition of 60 μM sample to hIR contacted with0.3 nM insulin resulted in an increase in phosphorylation ofapproximately three-fold and to the maximal level shown by insulinstimulation at higher concentrations. The EC₅₀ for this effect(enhancing insulin stimulation) was shown in additional experiments tobe approximately 20 μM of sample calculated as TER12. These results werealso confirmed by Western blot assay.

B. In an additional demonstration of the activity of a component in thesample containing TER12, the artificial substrate, poly(Glu₄ Tyr) wasused as the substrate for phosphorylation. Incorporation of labeledphosphate from γ-labeled ATP was measured following activation ofreceptor prepared as in Paragraph A. About 20 μM of sample calculated asTER12 provided 75% of maximal insulin-stimulated activity; it alsoenhanced the ability of 0.5 nM and 5.0 nM insulin to effectphosphorylation; 0.5 nM insulin alone showed 60% maximalphosphorylation; addition of 20 μM of the TER12 sample increased this to120%; in the presence of 5 nM insulin phosphorylation rose from 95% ofmaximum to 140%.

C. When tested with respect to insulin receptor agonist activity onwhole cells, i.e., on the human lymphocytic cell line IM-9, the samplecontaining TER12 retained its ability to stimulate the receptor. In thisassay, 2×10⁷ cells were treated with and without this sample and withand without insulin for 5 minutes, followed by three washes in isotonicmedium to remove the sample containing TER12. The cells were then lysedin 0.5% Tween 20 and lysates analyzed in an ELISA assay as described inParagraph A, without the steps of incubation with ATP. After 5 minutesexposure to sample containing 20 μM TER12, basal insulin receptor kinaseactivity was increased two-fold and insulin stimulated insulin receptorkinase activity was increased five-fold.

D. The assay described in paragraph B was conducted by substituting, forthe human insulin receptor, a recombinantly produced β chain lacking theinsulin-binding domain (supplied by Stratagene, Inc.). The ability ofthis kinase to phosphorylate a substrate peptide (Raytide from OncogeneSciences) is stimulated by TER12 at 25 μM. (In addition, a knowninhibitor believed to act at the ATP site on the kinase also inhibitsthis modified form of the receptor.)

E. Insulin is able to induce the differentiation of 3T3-L1 fibroblastcells to an adipocyte-like morphology as measured by Oil Red O uptake.The sample containing TER12 alone does not appear to effectdifferentiation; however, at a concentration of 20 μM it enhances thedifferentiating effect of insulin. This activity is similar to thatexhibited by pioglitazone described above. Insulin also enhances glucosetransport in this cell line. Again, the sample alone failed to stimulateglucose transport significantly, but enhanced the ability of insulin todo so.

EXAMPLE 2 Additional Compounds with TER12-Like Activity

Using substructure searching based on the TER12 molecule, 42 candidatecompounds were obtained and assayed according to the procedure ofExample 1.

A sample containing TER3938, shown in FIG. 2B, also showed agonistactivity. TER3938, shown in FIG. 2B and known as Direct Yellow No. 27,showed an EC₅₀ of 8 μM in this in vitro assay; it also enhanced theactivity of insulin in stimulating autophosphorylation of insulinreceptor on intact IM-9 cells. In addition, a sample containing TER3935,shown in FIG. 2C, was active in the IR kinase assay.

EXAMPLE 3 Identification of an Active Component of TER12 andTER3938-Containing Samples

TER12 was synthesized by the reaction scheme shown in FIG. 3. TER12synthesized using this scheme, and TER12 when extensively purified fromcommercial sources were active in the assays set forth in Example 1.

In addition, the sample containing TER3938, also obtained fromcommercial sources, when purified to 95% purity by reverse-phase HPLC,retained its activity; however, when this sample was washed with aqueoussodium carbonate, the insoluble compound shown in FIG. 2B as TER3938 wasless active in the IR kinase assay; the aqueous layer, however, retainedfull activity. These results led to the conclusion that some of theactivity shown in samples purportedly containing only TER12 and TER3938was due to a minor component. This minor component was postulated to beComponent A, which has the formula shown in FIG. 2F. Component A,obtained from commercial sources, was purified by C-18 reverse-phasepreparative HPLC and retained its activity in the IR kinase assay.Component A was subsequently demonstrated to be a minor component insamples containing both TER12 and TER3938. No Component A was found inTER3935 which is active after extensive purification.

Component A, purified from a commercially supplied sample, enhancesglucose uptake in differentiated 3T3-L1 cells, and the activity is notdependent on the presence of insulin. It is, however, dependent on theactivity of PI-3 kinase, confirming that the glucose uptake is mediatedvia the insulin signaling pathway. The ability of 16 μg/mlconcentrations of Component A to enhance glucose uptake at variousinsulin concentrations is shown in FIG. 4.

In the assay, 3T3-L1 pre-adipocytes were induced to differentiate intoadipocyte morphology using standard protocols. Five days afterinduction, the cells were treated with 16 μg/ml of Component A in thepresence of various levels of insulin for 30 minutes.

Glucose uptake was measured using ¹⁴ C glucose as label. As shown, 16μg/ml of Component A alone effects uptake at approximately the levelshown by 100 nM concentrations of insulin in the presence of thisconcentration of Component A.

EXAMPLE 4 Additional Compounds Related to TER3935

An additional compound with a structure regioisomeric to that ofTER3935, TER116998, was isolated by preparative reverse-phasechromatography from the reaction mixture produced by the syntheticscheme shown in FIG. 5. Spectral data confirm that the isolated compoundwas of the formula shown in FIG. 2D.

TER16998 activates the insulin receptor kinase directly, enhancesautophosphorylation and substrate phosphorylation mediated through theinsulin receptor, potentiates glucose transport and lowers blood glucosein the db/db mouse model of diabetes. These results were obtained asfollows:

The assay described in Example 1, paragraph A, was conducted with acontrol lacking any additions, in the presence of insulin alone at 1 nM,in the presence of TER16998 at 2 μM, and in the presence of acombination of these components at the stated concentrations. As shownin FIG. 6, TER16998 alone is able to activate autophosphorylation of thereceptor at this concentration, as well as to potentiate the effect ofinsulin.

In addition, in an assay for glucose uptake by 3T3-L1 adipocytes,described in Example 3, TER16998 produced an acute effect sensitizingthe cells to insulin. This was inhibited, as expected, by 5 μMwortmannin which inhibits PI-3 kinase, confirming that TER16998 exertsits effect through the insulin-signaling pathway. These results areshown in FIG. 7. As shown, 40 μM of TER16998 potentiates the effect ofinsulin at a range of concentrations.

Significantly, TER16998 was not able to stimulate the phosphorylationactivity of epidermal growth factor receptor in an EGF receptor kinaseassay.

The effect of TER16998 , of Component A, and of insulin on thedistribution of the Glut4 transporter in 3T3-L1 adipocytes wasdetermined by incubating the cells for 15 minutes with insulin or one ofthese compounds, after which the cells were fixed and stained with ananti-Glut4 antibody followed by FITC-conjugated secondary antibody. Theresults were visualized under a fluorescent microscope. The resultsshowed that insulin and Component A produce a dramatic redistribution ofGlut4 to the membrane surfaces whereas in untreated cells a diffusepattern is obtained. TER16998 has a similar effect but less dramaticthan that of insulin or Component A.

EXAMPLE 5 Effect of TER16998 in Diabetic Mice

Mice which are standard models of Type II diabetes, db/db mice, wereadministered TER16998 at 10 mg/kg and 40 mg/kg, or a vehicle as acontrol. FIG. 8 shows the effect of this compound on the concentrationof glucose in the blood of these animals. As shown in FIG. 8, 10 mg/kgto some extent and 40 mg/kg to an appreciable extent decrease bloodglucose over a period of 24 hours from the time of administration.

EXAMPLE 6 Synthesis of Invention Compounds

The polymeric aromatic compounds of the invention are synthesized usingeither solution-phase or solid-phase-based syntheses. For solid-phasesynthesis, the monomers are, for example, coupled to a phenolic resin ofthe formula 10 shown in FIG. 9. This phenolic resin is prepared byoxidation of the corresponding boronic ester polystyrene resin describedby Farrall, M. J. and Frechet, J. M. J. J Org Chem (1976) 41:3877. Thephenolic resin is condensed with an initial monomer, 11, as shown. Thecondensation product, 12, is treated with N-butyl lithium to replace thebromonium ion with lithium and this intermediate is condensed with amonomer, for example, of the formula 13, to provide the solid-supporteddimer, 14. The linking --CHOH-- can be, if desired, reduced to --CH₂ --.Subsequent condensations in the presence of formaldehyde andconcentrated sulfuric acid result in polymers of the desired lengthwhich can then be removed from the resin in sodium methoxide. Thisprocess is outlined in FIG. 9.

The synthesis can be varied by altering the nature of the monomer ordimer added to the supported starting group.

Synthesis of suitable oligomers is also conducted in solution phase. Inone set of reactions, the synthesis is a variant of that described byArduini, A. et al. Tetrahedron (1990) 46:3607-3613. Intermediate dimersor trimers are obtained by condensing an aromatic aldehyde with anaromatic bromide and further condensation is effected in the presence offormaldehyde and sulfuric acid. Dimers shown as formulas 18, 19 and 20,which contain the proton-accepting substituent in all possibleconfigurations can be obtained by condensation of the appropriatenaphthyl lithium with a naphthyl formyl derivative obtained by treatingthe naphthyl lithium with dimethyl formamide (DMF). If desired,compounds 18, 19 and 20 can be reduced with HSiEt₃ /TFA to thecorresponding methylene-bridged dinaphthylenes. ##STR6## The dibromodimer of the form 23 is obtained as described by Arduini, A. (supra) bycondensing the corresponding naphthyl bromide with formaldehyde in thepresence of sulfuric acid. ##STR7##

The corresponding trimers can be obtained by treating suchbromo-substituted dimers with N-butyl lithium and then with an aldehydeof the formula 21 or 22. ##STR8##

The oligomers may also be extended by coupling naphthyl or otheraromatic moieties with dialdehydes, for example,4,4'-biphenyldicarboxaldehyde or terephthaldehyde.

By appropriate choice of the position of the proton-acceptingsubstituent A, and of the reactive substituents on the aromaticmoieties, the desired oligomers may be synthesized.

As set forth above, the polymer of the formula ##STR9## is active in theinsulin receptor kinase assay described above and exhibits the abilityto potentiate insulin activation and glucose uptake.

We claim:
 1. A method to modulate the kinase activity of insulinreceptor which method comprises contacting said insulin receptor or thekinase portion thereof with a compound of the formula ##STR10## whereineach Ar is independently an aromatic moiety, each A is independently aproton-accepting substituent;each R is independently a noninterferingsubstituent; m is 0 or 1; n is 4-6; and each linker is independently anisostere of --CH₂ --, --CH═CH-- or --NHCO--.
 2. The method of claim 1wherein each A is independently --SO₃ X or --COOX wherein X is H or acation.
 3. The method of claim 1 wherein each R is independently alkyl(1-6C).
 4. The method of claim 1 wherein all m are
 0. 5. The method ofclaim 1 wherein said compound is of the formula ##STR11## wherein n is4-6 and A is as defined in claim
 1. 6. A method to potentiate theinsulin activation of insulin receptor which method comprises contactingsaid insulin receptor or the kinase portion thereof with insulin andwith a compound of the formula ##STR12## wherein each Ar isindependently an aromatic moiety; each A is independently aproton-accepting substituent;each R is independently a noninterferingsubstituent; m is 0 or 1; n is 4-6; and each linker is independently anisostere of --CH₂ --, --CH═CH-- or --NCHO--; said compound provided inan amount effective to potentiate said insulin activation.
 7. The methodof claim 6 wherein each A is independently --SO₃ X or --COOX wherein Xis H or a cation.
 8. The method of claim 6 wherein each R isindependently alkyl(1-6C).
 9. The method of claim 6 wherein all m are 0.10. The method of claim 6 wherein said compound is of the formula##STR13## wherein n is 4-6 and A is as defined in claim 1.